Fig 1.
CR-AnxA12-48 is resistant to neutrophil mediated cleavage and selectively binds the ALX receptor.
(A) Aminoacid sequences for CR-AnxA12–50 and CR-AnxA12–48 (B) Plots depicting the amount of peptide remaining at the indicated intervals in incubations of human neutrophil with CR-AnxA12–50 or CR-AnxA12–48. Results are mean ± SEM of n = 4 distinct incubations with different neutrophil preparations. *p < 0.05 versus AnxA12–48 incubations. (C,D) Binding affinity of CR-AnxA12–48 to (C) human ALX-overexpressing and (D) FPR1-overexpressing HEK cells was assessed in a competitive binding assay using [125I]–Ac2-26. (E) Agonist activity of CR-AnxA12–48 was investigated using β-arrestin stably expressing FPR2/ALX. Results for C-E are mean ± SEM of 3–4 independent experiments.
Fig 2.
Anti-inflammatory and pro-resolving actions of CR-AnxA12-48 with mouse and human leukocytes.
(A) Plots depicting phagocytosis of apoptotic human neutrophils (efferocytosis) by macrophages incubated with or without the indicated concentrations of CR-AnxA12–50 and CR-AnxA12–48. (B) Bar graphs depicting phagocytosis of apoptotic human neutrophils (efferocytosis) by bone marrow derived macrophages from Alx/Fp2/3-/- mice incubated with or without the indicated concentrations of CR-AnxA12–48. Results are mean ± SEM. n = 4 mice per group per time point; *p < 0.05, **p < 0.01, versus vehicle group. (C) Plots depicting the number of human neutrophils interacting with activated human endothelial cells incubated with or without the indicated concentrations of CR-AnxA12–48.. (D) Bar graphs illustrating the number of neutrophils interacting with activated endothelial cells. Results for C,D are mean ± SEM of n = 4 independent cellular preparations *p < 0.05 versus PBS, **p < 0.01 versus vehicle control.
Fig 3.
CR-AnxA12–48 regulated neutrophil recruitment in vivo.
Graphs illustrate (A) total leukocyte numbers and (B) the number of Ly6G+ cells quantified using light microscopy and flow cytometry (see Materials and Methods for details). Results are mean ± SEM of n = 4 mice per group; *p < 0.05, **p < 0.01, versus vehicle group.
Fig 4.
CR-AnxA12–48 displays cardioprotective actions in murine ischemia reperfusion mediated injury.
Graphs depict (A) Relative area at risk to total ventricle size in each group (B-C) Quantitative analyses for tissue damage expressed as (B) infarct size/area at risk and (C) infarct size/left ventricle. (D) Plasma CCL5 concentrations. Results are mean ± SEM of n = 6 mice per group. **p < 0.01 versus Vehicle group.